Proteomics Shared Resource Description

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The University of Arizona Cancer Center Proteomics Shared Resource operates under the administrative umbrella of the Arizona Proteomics Consortium (Dr. George Tsaprailis, Director). The Consortium provides Proteomics Resources to Arizona Cancer Center Members and Staff and includes one facility on the University’s Main Campus (2,000 sq. ft.), and two on the Arizona Health Sciences Center (AHSC) campus: in the BIO5 Institute Keating Bioresearch Building (3,245 sq.ft.); and in the College of Pharmacy. This third location is within easy walking distance of the University of Arizona Cancer Center and offers an additional 1,800 sq.ft. of laboratory space which can be expanded.

The University of Arizona Cancer Center Proteomics Shared Resource is staffed by Dr. Linda Breci (Associate Director of the Arizona Proteomics Consortium), Dr. Cynthia David (Assistant Staff Scientist), Yelena Feinstein (Research Specialist), and Scott DeRigne, an Instrumentation Specialist who holds an BS in Electrical Engineering. The Instrumentation Specialist is responsible for routine maintenance and repair of all mass spectrometry hardware and related proteomics auxiliary equipment. All personnel under the supervision of the Director are responsible for implementing current and designing new proteomic capabilities for users.

The Consortium houses three working quadrupole ion trap mass spectrometers (one Thermo Finnigan LCQ DECA XP+ and two LTQs; two older ion traps have been retired due to lack of parts and support from manufacturer), each complete with nano HPLCs. These nano HPLCs are from Michrom Bioresources (Paradigm MS4 HPLC on one of the LTQ systems), and ThermoFinnigan (Surveyor HPLCs on the LCQ DECA XP+ and second LTQ). The ion traps are also equipped with autosamplers. All nano HPLCs are able to carry solvent at 300-400 nL/min into the various mass spectrometers. All ion traps are equipped with custom-built nanoelectrospray sources based on the original designs of Dr. John Yates III at the University of Washington. With the exception of one nanospray source that was produced in the University of Washington machine shop, the rest were built by the University of Arizona machine shop. The Michrom Paradigm MS4 HPLC is a quaternary nano-HPLC systems with two switching valves for operation in tandem LC (ion exchange-reverse phase) mode for analyses of complex peptide mixtures. Both LTQs can also perform LC-LC-MS/MS on an as-needed basis. Separation of protein digests for LC-MS/MS is achieved using fused silica capillaries (100 micron I.D. x 360 micron O.D) that are pulled  to an opening of 3-5 microns using a laser capillary puller from Sutter Instruments (P-2000 Microcapillary puller) and packed in-house with 7 cm of Vydac C18 material. All systems are configured for automated operation and data processing under computer control.

Instrumentation grant awards to Dr. Tsaprailis have allowed for additional state-of-the-art instrumentation including the acquisition of a hybrid triple quadrupole linear ion trap from Applied Biosystems (4000 QTRAP), which, along with a Proxeon Easy nLC and an ABI nanospray source, is dedicated to more comprehensive protein characterization experiments such as targeted MS/MS and post-translational modification mapping owing to specific triple-quadrupole type of scanning abilities (MRMs). A new Chipmate nanoESI source from Advion was recently purchased for the 4000 QTRAP to facilitate higher throughput sample analysis. The most recent hardware acquisitions to the Shared Resource are a Thermo Scientific LTQ Orbitrap Velos mass spectrometer with Advion Nanomate ESI source, and a Proxeon nanoLC, made possible through a High End Instrumentation Grant from NIH/NCRR to Dr. Tsaprailis in the Spring of 2010. Along with collision-activated dissociation, this mass spectrometer features two alternative activation capabilities: HCD and ETD, the latter can be used to map labile posttranslational modifications. Top-down sequencing of small-to-medium proteins is also possible on the LTQ Orbitrap Velos.

At the College of Pharmacy facility, label-free protein expression measurements in the liquid phase following protein digestion are possible using a Waters Q-TOF Premier mass spectrometer and nanoACQUITY HPLC system. This facility also houses a research grade MALDI-TOF mass spectrometer from ABI (AB Voyager DE-STR, Framingham, MA) for tissue imaging and also routine mass mapping and molecular weight measurements. A FTMS and a MALDI TOF-TOF from Bruker are also available for use at the UA Main Campus facility.

Proteomics software for protein identification using LC-MS/MS data include TurboSEQUEST (the Consortium has five licensed versions running on five separate web servers as well as a Linux Sorcerer 2 Sequest search engine), X!Tandem, and a licensed version of MASCOT. Three licensed versions of Scaffold are also used for protein identification from peptide MS/MS data; these run on dedicated Pentium PCs. Another MS/MS data search engine in use is OMSSA available through NCBI. The Arizona Proteomics Consortium has approximately 30 Pentium PCs and various laptops, used as stand-alone devices or as part of the instrument hardware control.

Other Proteomics-related equipment available include a Thermo Separation Products Quaternary solvent delivery system, and a second microbore HPLC from Michrom Bioresources for protein and peptide purification (Paradigm MS4B). A GE Healthcare AKTA FPLC is used for various modes of protein chromatography, including an affinity column to remove abundant proteins from biological samples such as sera and plasma to allow discovery of less abundant, and possibly more clinically interesting, proteins The Proteomics Shared Resource is also equipped with BioRad Protean, MiniProtean, and Criterion 1D- and 2D-electrophoresis setups. Gel imaging and spot picking capabilities are made possible using a Genomic Solutions UV-vis gel imager/spot picker (ProPic II). There is also an accompanying digester, and a MALDI-target spotter from Genomic Solutions. These instruments provide higher-throughput preparation for MS analyses of gel and solution-based protein/peptide samples. A Perkin Elmer liquid handling robot for automated digestion of samples is also available.

The Proteomics Shared Resource also offers macromolecule interaction characterization services using a Biacore T100 surface plasmon resonance (SPR) instrument. The T100 detects interactions between proteins, peptides, nucleic acids, lipids, carbohydrates, small molecules, viruses, and even whole cells in real time without the use of labels. Affinity KDs, kon, koff rates, and equilbrium KDs can be determined in the mM to pM range. Also, because analysis temperature can be varied between 4-45°C, thermodyamic studies are possible. A PhD Staff Scientist is dedicated to method development, instrument operation, and data analysis.